Over the past decade, genome-wide association studies and comprehensive whole-genome sequencing analyses have uncovered both common and rare variants that are associated with a wide range of disease- related traits. The majority of these associated variants lie in intergenic regions, and it has been suggested that they modulate the expression of individual genes. Indeed, genetic analyses have identified a large number of expression quantitative trait loci adjacent to the affected genes (cis-eQTL), and many sequence variants are strongly associated with both the expression of individual genes and disease-related traits. However, deciphering the underlying molecular mechanisms has been challenging. Traditional laboratory approaches, such as luciferase reporter constructs or electrophoretic mobility shift assays, as well as recent chromatin analyses (DNase hypersensitivity mapping, ATAC-Seq) clearly suggest a functional impact for disease- associated promoter variants, but no effective methods exist to identify the regulatory proteins binding to these variant sites and mediating their effect on the regulation of gene expression. Therefore, alternative approaches are required to more efficiently identify these unknown regulatory proteins whose promoter binding and interaction is affected by eQTL variants. We recently developed a novel approach, Hybridization Capture of Chromatin-Associated Proteins for Proteomics (HyCCAPP), that allows the identification of all proteins bound to a specific target chromatin region by mass spectrometry. We propose to adapt the HyCCAPP approach for the analysis of luciferase reporter plasmid constructs commonly used to assess the impact of sequence variants on promoter activity, and apply the technology to the analysis of selected eQTL promoter variants. We hypothesize that the HyCCAPP technology will uncover novel regulatory proteins mediating the effect of promoter variants on gene expression, revealing potentially novel molecular mechanisms underlying eQTLs. We will pursue three Specific Aims: 1) optimize HyCCAPP for the analysis of luciferase reporter plasmids, 2) analyze select promoter variants in eQTL regions using plasmid HyCCAPP, and 3) validate the impact of protein binding on gene expression in vivo in CRISPR-Cas9 edited cell lines. The technology development of HyCCAPP to target luciferase reporter plasmids, and a proof-of-principle application to promoter variants associated with gene expression changes, will establish a powerful new and effective tool for the investigation of the mechanisms by which regulatory sequence variants alter binding of regulatory proteins. No other current technology allows the effective de novo identification of DNA binding proteins affected by sequence variants. The use of commonly used luciferase reporter plasmid approaches with the new HyCCAPP technology will help reveal new mechanisms of gene expression regulation contributing to the development and progression of human disorders.